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R W Alexander

Publications and source records attributed to R W Alexander.

At least 91 records · Page 5Linked to original sources
PubMed1994

Epitope mapping of the alpha-chain of the insulin-like growth factor I receptor using antipeptide antibodies.

Insulin-like growth factor I (IGF I) is an important mitogen for vascular smooth muscle cells (VSMC). The IGF I receptor (IGF IR) is a heterotetramer composed of two cross-linked extracellular alpha-chains and two membrane-spanning beta-chains that contain a tyrosine-kinase domain. It has a high degree of sequence similarity to the insulin receptor (IR), and the putative ligand-specific binding site has been localized to a cysteine-rich region (CRR) of the alpha-chain. To obtain insights into antigenic determinants of the IGF IR, we raised a panel of site-specific polyclonal antibodies against short peptide sequences N-terminal to and within the CRR. Several antibodies raised against linear epitopes within the CRR bound to solubilized and native rat and human IGF IR by ELISA, did not cross-react with IR, but unexpectedly failed to inhibit 125I-IGF I binding. A polyclonal antibody directed against a 48-amino acid synthetic peptide, corresponding to a region of the CRR postulated to be essential for ligand binding, failed to react with either solubilized, reduced or intact IGF IR. Three antibodies specific for the N-terminus of the alpha-chain reacted with solubilized and native IGF IR. One of these, RAB 6, directed against amino acids 38-44 of the IGF IR, inhibited 125I-IGF I binding to rat aortic smooth muscle cells (RASM) and to IGF IR/3T3 cells (overexpressing human IGF IR) by up to 45%. Immunohistochemical analysis revealed strong IGF IR staining in the medial smooth muscle cell layer of rat aorta. These findings are consistent with a model wherein conformational epitopes within the CRR and linear epitopes within the N-terminus of the alpha-chain contribute to the IGF I binding pocket. These antibodies should provide a valuable tool to study structure-function relationships and in vivo regulation of the IGF IR.

Amino Acid Sequence↗
PubMed1994

Agonist-induced phosphorylation of the vascular type 1 angiotensin II receptor.

Agonist-induced receptor phosphorylation plays a role in transmembrane signal transduction systems. Although the cDNA for the rat vascular type 1 angiotensin II receptor (AT1AR) encodes a G protein-coupled receptor with several potential phosphorylation sites for serine/threonine and tyrosine kinases, little is known about the phosphorylation of this receptor. The aim of this study was to determine the effects of angiotensin II (Ang II) on phosphorylation of the AT1AR in rat aortic vascular smooth muscle cells. Using [32P]orthophosphate-labeled cells, immunoprecipitates with anti-AT1AR antibody revealed a labeled band of molecular weight 52 kD, corresponding to the Ang II receptor. Ang II induced a rapid and significant increase in phosphorylation of the Ang II receptor, with a peak at 20 minutes. Phosphoamino acid analysis showed that the major phosphoamino acid is serine, in both the basal and Ang II-stimulated states. Constitutive and agonist-stimulated tyrosine phosphorylation is also observed to a lesser extent. Immunoblotting of anti-phosphotyrosine immunoprecipitates with anti-AT1AR antibody showed that Ang II caused a delayed tyrosine phosphorylation of the receptor with a peak at 20 minutes in a dose-dependent manner. Forskolin increased total phosphorylation of AT1AR but had no effect on tyrosine phosphorylation. Neither phorbol 12-myristate-13-acetate nor ionomycin altered receptor phosphorylation. These findings suggest that Ang II induces the phosphorylation of its own G protein-coupled receptor through both serine and tyrosine kinases and raise the possibility that phosphorylation of the AT1AR is an important regulator of receptor function.

Angiotensin II↗
PubMed1994

Angiotensin II stimulates NADH and NADPH oxidase activity in cultured vascular smooth muscle cells.

The signaling pathways involved in the long-term metabolic effects of angiotensin II (Ang II) in vascular smooth muscle cells are incompletely understood but include the generation of molecules likely to affect oxidase activity. We examined the ability of Ang II to stimulate superoxide anion formation and investigated the identity of the oxidases responsible for its production. Treatment of vascular smooth muscle cells with Ang II for 4 to 6 hours caused a 2.7 +/- 0.4-fold increase in intracellular superoxide anion formation as detected by lucigenin assay. This superoxide appeared to result from activation of both the NADPH and NADH oxidases. NADPH oxidase activity increased from 3.23 +/- 0.61 to 11.80 +/- 1.72 nmol O2-/min per milligram protein after 4 hours of Ang II, whereas NADH oxidase activity increased from 16.76 +/- 2.13 to 45.00 +/- 4.57 nmol O2-/min per milligram protein. The NADPH oxidase activity was stimulated by exogenous phosphatidic and arachidonic acids and was partially inhibited by the specific inhibitor diphenylene iodinium. NADH oxidase activity was increased by arachidonic and linoleic acids, was insensitive to exogenous phosphatidic acid, and was inhibited by high concentrations of quinacrine. Both of these oxidases appear to reside in the plasma membrane, on the basis of migration of the activity after cellular fractionation and their apparent insensitivity to the mitochondrial poison KCN. These observations suggest that Ang II specifically activates enzyme systems that promote superoxide generation and raise the possibility that these pathways function as second messengers for long-term responses, such as hypertrophy or hyperplasia.

Acridines↗
PubMed1993

Mapping the central fold of tRNA2(fMet) in the P site of the Escherichia coli ribosome.

4-Thiouridine (s4U), a photoreactive analog of uridine, was randomly incorporated into tRNA2(fMet) precursor molecules by transcription with T7 RNA polymerase. The s4U-containing transcripts were trimmed at their 5'-ends with RNase P RNA to yield mature tRNA2(fMet). The photoreactive tRNA2(fMet) derivatives were aminoacylated and bound to the P site of 70S ribosomes from Escherichia coli in the presence of a poly(A,G,U) template. Irradiation of the complexes at 300 nm resulted in the covalent cross-linking of tRNA2(fMet) to ribosomal proteins and rRNAs within both the 50S and 30S subunits. The labeled proteins were identified as L1, L27, and S19. 50S-subunit proteins L1 and L27 were attached to nucleotide U17 or U17.1 within the D loop of tRNA2(fMet), whereas 30S-subunit protein S19 was cross-linked to nucleotide U47 in the variable loop. Both of these sites occur in or near the central fold of the tRNA. These results permit us to map the D loop of P site-bound tRNA to the region between the central protuberance and the L1 ridge on the 50S ribosomal subunit, while the variable loop can be placed above the cleft on the head of the 30S subunit.

Base Sequence↗
PubMed1993

Phosphatidylcholine is a major source of phosphatidic acid and diacylglycerol in angiotensin II-stimulated vascular smooth-muscle cells.

In cultured vascular smooth-muscle cells, angiotensin II produces a sustained formation of diacylglycerol (DG) and phosphatidic acid (PtdOH). Since the fatty acid composition of these molecules is likely to determine their efficacy as second messengers, it is important to ascertain the phospholipid precursors and the biochemical pathways from which they are produced. Our experiments suggest that phospholipase D (PLD)-mediated phosphatidylcholine (PtdCho) hydrolysis is the major source of both DG and PtdOH during the late signalling phase. First, in cells labelled with [3H]myristate, which preferentially labels PtdCho, formation of [3H]PtdOH precedes formation of [3H]DG. Second, in contrast with phospholipase C (PLC) activation, DG mass accumulation is dependent on extracellular Ca2+. Similarly, DG mass accumulation is not attenuated by protein kinase C activation, which we have previously shown to inhibit the phosphoinositide-specific PLC. Third, the fatty acid composition of late-phase DG and PtdOH more closely resembles that of PtdCho than that of phosphatidylinositol. Finally, in cells labelled for a short time with [3H]glycerol, the radioactivity incorporated into [3H]DG and PtdOH was greater than that incorporated into PtdIns, but not into PtdCho. We found no evidence that synthesis de novo or phosphatidylethanolamine breakdown contributes to sustained DG and PtdOH formation. Thus, in angiotensin II-stimulated cultured vascular smooth-muscle cells, PLD-mediated PtdCho hydrolysis is the major source of sustained DG and PtdOH, whereas phosphoinositide breakdown is a minor contributor. Furthermore, PtdOH phosphohydrolase, which determines the relative levels of DG and PtdOH, appears to be regulated by protein kinase C. These results have important implications for the role of these second messengers in growth and contraction.

Angiotensin II↗
PubMed1993

Hydrogen peroxide-induced c-fos expression is mediated by arachidonic acid release: role of protein kinase C.

We found previously that stimulation of c-fos and c-myc mRNA expression are early events in hydrogen peroxide-induced growth in rat aortic smooth muscle (RASM) cells. In the present study, we investigated the role of phospholipase A2 (PLA2) and protein kinase C (PKC) in mediating hydrogen peroxide-induced c-fos mRNA expression in RASM cells. Mepacrine and p-bromophenacylbromide, potent inhibitors of PLA2 activity, blocked hydrogen peroxide-induced c-fos mRNA expression. Arachidonic acid, a product of PLA2 activity, stimulated the expression of c-fos mRNA with a time course similar to that of hydrogen peroxide. PKC down-regulation attenuated both hydrogen peroxide and arachidonic acid-induced c-fos mRNA expression by 50%. Nordihydroguaiaretic acid (a lipoxygenase-cytochrome P450 monooxygenase inhibitor) significantly inhibited both hydrogen peroxide and arachidonic acid-induced c-fos mRNA expression, whereas indomethacin (a cyclooxygenase inhibitor) had no effect. Together, these findings indicate that 1) hydrogen peroxide-induced c-fos mRNA expression is mediated by PLA2-dependent arachidonic acid release, 2) both PKC-dependent and independent mechanisms are involved in hydrogen peroxide-induced expression of c-fos mRNA and 3) arachidonic acid metabolism via the lipoxygenase-cytochrome P450 monooxygenase pathway appears to be required for hydrogen peroxide-induced expression of c-fos mRNA.

Animals↗
PubMed1993

Polarized secretion of IGF-I and IGF-I binding protein activity by cultured aortic endothelial cells.

Insulin-like growth factor-I (IGF-I) secretion by the vascular endothelium has been proposed to play a role in the regulation of vascular smooth muscle cell proliferation. Because vascular smooth muscle cells are adjacent to the abluminal surface of the endothelium, we tested the hypothesis that secretion of IGF-I by endothelial cells is polarized. Porcine aortic endothelial cells were cultured on permeable membranes and IGF-I measured by radioimmunoassay. Basal secretion exceeded apical secretion by a ratio of 2.3 +/- 0.2:1.0 (P < 0.05). We also identified 35 kDa IGF-I binding protein activity that is preferentially secreted on the basal surface of endothelial cells. We conclude that both IGF-I and IGF-I binding protein activity secretion by endothelial cells is polarized towards the basal surface of the endothelium. A polarized secretion mechanism for IGF-I may be of importance in the normal growth and differentiation of the vasculature as well as in the development of vascular pathology.

Animals↗
PubMed1993

The dysfunctional endothelium in heart failure.

Through release of paracrine and autocrine substances, the vascular endothelium exerts a profound influence on the contractile and growth state of underlying vascular smooth muscle. In heart failure, there is now compelling evidence that endothelium-mediated relaxation is attenuated in response to muscarinic stimulation in both the coronary and peripheral circulation of humans. This impaired response is present regardless of the etiology of heart failure. The mechanisms, significance and etiologic importance of this endothelial defect are not yet understood. Possibilities include 1) alterations in endothelial cell surface receptors or abnormalities of postreceptor signal transduction; 2) abnormalities of endothelium-derived relaxing factor production or release; 3) rapid inactivation of endothelium-derived relaxing factor; and 4) an increase in endothelium-derived contracting factor production and activity in heart failure. We currently have little understanding of the mechanisms accounting for this dysfunctional state of the endothelium. Future research efforts should be directed toward an understanding of these mechanisms.

Animals↗
PubMed1993

Induction of cardiac insulin-like growth factor I gene expression in pressure overload hypertrophy.

Molecular mechanisms regulating the cardiac hypertrophic response to increased hemodynamic load are understood poorly. Insulin-like growth factor I (IGF I) is a mitogen that is thought to play a key role in pre- and postnatal growth. To investigate a possible role of IGF I in the cardiac response to pressure overload, rats underwent banding of the ascending aorta immediately above the aortic valve using a hemoclip, or a sham procedure. An analysis of left-ventricular RNA by Northern hybridization using a 32P-labeled IGF I cDNA revealed four messenger ribonucleic acid transcripts of 7.6, 4.6, 1.7, and 0.9 to 1.2 Kb. Insulin-like growth factor I messenger ribonucleic acid was quantitated by ribonuclease protection assays using a rat exon 3 riboprobe. There was a sustained increase in IGF I mRNA levels that correlated temporally with the development of left ventricular hypertrophy. These results indicate that left ventricular pressure overload is associated with an induction of cardiac IGF I gene expression. Insulin-like growth factor I may play a role in the response to increases in wall stress and likely contribute to cardiac hypertrophy.

Animals↗
PubMed1993

Hemodynamics and vascular endothelial biology.

The vascular endothelium, as the interface between flowing blood and the underlying vessel wall, not only resides in a hemodynamic environment, but also is a mediator of flow-related influences. In recent years, particularly with the advent of cell culture studies, much has been learned about the effects of this mechanical environment. This brief review focuses on these more recent cell culture studies, emphasizing the effects of flow and the associated shear stress on vascular endothelial biology. Included are endothelial cell shape and orientation, cytoskeletal components, endocytosis, proliferation, and signal recognition/transduction mechanisms. Of particular interest are mechanical effects on the release of vasoactive substances, and recent studies of prostacyclin, endothelin, and nitric oxide are discussed. Through this review it will be demonstrated that flow has a major influence on the synthesis and release of such substances.

Animals↗
PubMed1993

Autocrine control of wound repair by insulin-like growth factor I in cultured endothelial cells.

The repair process of the vascular endothelium is modulated by growth factors from both endogenous (within the vessel wall) and exogenous (blood borne) sources. We utilized a tissue culture model of endothelial wounding to gain further insight into the potential autocrine control of proliferation during wound repair. Cultured porcine aortic endothelial monolayers were mechanically wounded by passing a 7-mm sterile glass rod over the surface of the culture. Proliferation at the wound edge was quantified using [3H]thymidine autoradiography. In wounded cultures incubated in media supplemented with 10% fetal calf serum, 81 +/- 2% of the nuclei at the wound edge were labeled. When the cultures were incubated in serum-free media, proliferation at the wound edge was only slightly diminished with 65 +/- 3% (P < 0.05) of the cells labeled. These findings raise the possibility that there is a significant contribution from autocrine growth factors to endothelial wound repair. To evaluate the potential role of insulin-like growth factor I (IGF-I) in the wound repair process, we used a radioimmunoassay to measure IGF-I secretion. Wounded cultures exhibited a 187 +/- 58% increase in IGF-I production when compared with nonwounded cultures (P < 0.05). To determine the extent to which endogenous IGF-I mediates the proliferative response of endothelial cell monolayers to wounding, wounded cultures were incubated with inactivating concentrations of IGF-I antibody. When IGF-I antibody was present in the culture media, only 26 +/- 3% of the nuclei at the wound edge were labeled with [3H]thymidine (P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Fluid shear stress stimulates platelet-derived growth factor expression in endothelial cells.

Fluid flow and the associated shear stress play a critical role in vascular growth and remodeling. Recent data suggest that increased endothelial cell expression of platelet-derived growth factor (PDGF) A- and B-chain by flow may participate in these events. In the present study, we examined the mechanism for flow-induced PDGF expression, focusing on protein kinase C (PKC). Bovine aortic endothelial cells were exposed to flow (shear stress = 30 dyn/cm2) in a parallel-plate flow chamber. Increases in PDGF B-chain, but not PDGF A-chain, were observed within 3 h, maximal within 6 h (13-fold increase), and sustained for 24 h. PKC appeared to be involved because phorbol 12-myristate 13-acetate induced PDGF B-chain mRNA. Activation of PKC alone, however, was insufficient to induce PDGF mRNA because the selective PKC activator, 1-oleoyl-2-acetyl-sn-glycerol, did not induce PDGF expression. A PKC-independent pathway was suggested by the fact that inhibition of PKC (downregulation with phorbol 12,13-dibutyrate or exposure to staurosporine) failed to block PMA or flow-induced PDGF B-chain expression. These results demonstrate flow-induced PDGF B-chain expression in endothelial cells that appears to be mediated, in part, by a PKC-independent pathway.

Animals↗
PubMed1993

Immunohistochemical localization of rat angiotensin II AT1 receptor.

To study receptors for angiotensin II, polyclonal rabbit anti-peptide antisera were prepared against the peptide QDDCPKAGRHC corresponding to amino acids 15-24 of the rat AT1A and AT1B receptors. Western analysis of rat tissues showed a major band of approximately 43 kDa. The antisera immunoprecipitated AT1-receptor protein produced in vitro. Immunohistochemical analysis of rat tissues showed intense staining of arterial and arteriolar smooth muscle. Other tissues that contained AT1-receptor protein included hepatocytes, the zona glomerulosa of the adrenal gland, and the smooth muscle of the bronchus, gut, ureter, and epididymis. In the kidney, intense staining was observed in all small arteries and arterioles. Both afferent and efferent arterioles contain approximately equal intensities of immunoreactive AT1 protein. The inner stripe of the outer medulla has a moderate level of receptors within thick ascending limb epithelium. Proximal tubular epithelium also expresses receptor protein. Glomerular immunoreactive AT1 protein is found within mesangial cells and varies in intensity among different rat strains. Lewis and Wistar rats demonstrated moderate glomerular staining, whereas the CD and Sprague-Dawley strains showed lesser levels of reactivity. The fact that glomerular mesangial cells are the primary locus of angiotensin II action within the glomerulus.

Amino Acid Sequence↗
PubMed1993

Hypertension and left ventricular hypertrophy are associated with impaired endothelium-mediated relaxation in human coronary resistance vessels.

BACKGROUND: Patients with hypertension and myocardial hypertrophy may have signs and symptoms of myocardial ischemia in the absence of obstructive coronary disease. Prior investigations have demonstrated impaired coronary flow reserve and have led to speculation that microvascular dysfunction might contribute to ischemia in these patients. Experimental studies have shown that the endothelium, an important regulator of microvascular tone, can be damaged by hypertension and is dysfunctional in cardiomyopathy. We hypothesized that endothelium-dependent vasodilation is impaired in the coronary microvasculature of patients with hypertension and ventricular hypertrophy. METHODS AND RESULTS: We studied coronary microvascular responses in 10 patients with left ventricular hypertrophy secondary to essential hypertension (HTN) (mean arterial pressure at catheterization, 151/94 mm Hg; mean posterior wall thickness, 1.4 +/- 0.1 cm) and nine normal control subjects with no history of hypertension (mean arterial pressure at catheterization, 128/75 mm Hg; mean posterior wall thickness, 1.0 +/- 0.02 cm) using the intracoronary Doppler catheter and quantitative angiography to assess changes in coronary blood flow (CBF). All patients had normal left ventricular systolic function. To assess microvascular endothelial function, we infused the endothelium-dependent vasodilator acetylcholine (10(-8)-10(-6) M) and the endothelium-independent vasodilator adenosine (10(-6)-10(-4) M) into the left anterior descending coronary artery. In response to acetylcholine, CBF increased only 32 +/- 25% in HTN patients, whereas CBF increased 192 +/- 39% in normal control subjects (p = 0.003). CBF increased 465 +/- 93% in HTN patients and 439 +/- 41% in normal control subjects in response to adenosine (p = NS). The proportion of coronary flow reserve attributable to endothelium-dependent dilation (obtained from peak acetylcholine/peak adenosine flow response) was 48 +/- 9% in normal control subjects but only 7 +/- 8% in HTN patients (p = 0.008). CONCLUSIONS: Endothelium-dependent vasodilation is markedly impaired in the coronary microvessels of patients with hypertension and ventricular hypertrophy. Loss of this vasodilator mechanism may contribute to disordered coronary flow regulation and the ischemic manifestations of hypertensive heart disease.

Acetylcholine↗
PubMed1993

Abdominal coarctation increases insulin-like growth factor I mRNA levels in rat aorta.

We have previously demonstrated specific insulin-like growth factor I (IGF I) mRNA transcripts in cultured endothelial and vascular smooth muscle cells and postulated an important role for IGF I in blood vessel growth responses. The purpose of this study was to characterize IGF I gene expression in a model of aortic coarctation hypertension in the rat. This high-renin model of hypertension is associated with hyperplastic vascular responses. Northern analysis of rat aorta demonstrated four specific IGF I mRNA transcripts sized 7.6, 4.6, 1.8, and 0.9-1.2 kb. Quantitation of aortic IGF I mRNA levels by solution hybridization/RNase protection assay demonstrated induction of IGF I transcripts in the hypertensive aorta; levels more than doubled at 7 days and were still significantly elevated 21 days after coarctation. In situ hybridization analysis indicated that IGF I transcripts were localized primarily to adventitial surfaces in normotensive aorta, with minimal signal detected over vascular cells. In hypertensive aortas, there was an increase in IGF I transcripts primarily over vascular smooth muscle cells. Thus, vascular IGF I gene expression is induced in this model of high-renin hypertension. IGF I may play an important role in autocrine/paracrine-mediated vessel wall remodeling in hypertension.

Animals↗